Osteosarcoma cells exhibit functional interactions with stromal cells, fostering a lung microenvironment conducive to the establishment of metastatic tumor cells.

BACKGROUND: Osteosarcoma (OS) stands out as the most common bone tumor, with approximately 20% of the patients receiving a diagnosis of metastatic OS at their initial assessment. A significant challenge lies in the frequent existence of undetected metastases during the initial diagnosis. Mesenchymal stem cells (MSCs) possess unique abilities that facilitate tumor growth, and their interaction with OS cells is crucial for metastatic spread. METHODS AND RESULTS: We demonstrated that, in vitro, MSCs exhibited a heightened migration response toward the secretome of non-metastatic OS cells. When challenged to a secretome derived from lungs preloaded with OS cells, MSCs exhibited greater migration toward lungs colonized with metastatic OS cells. Moreover, in vivo, MSCs displayed preferential migratory and homing behavior toward lungs colonized by metastatic OS cells. Metastatic OS cells, in turn, demonstrated an increased migratory response to the MSCs' secretome. This behavior was associated with heightened cathepsin D (CTSD) expression and the release of active metalloproteinase 2 (MMP2) by metastatic OS cells. CONCLUSIONS: Our assessment focused on two complementary tumor capabilities crucial to metastatic spread, emphasizing the significance of inherent cell features. The findings underscore the pivotal role of signaling integration within the niche, with a complex interplay of migratory responses among established OS cells in the lungs, prometastatic OS cells in the primary tumor, and circulating MSCs. Pulmonary metastases continue to be a significant factor contributing to OS mortality. Understanding these mechanisms and identifying differentially expressed genes is essential for pinpointing markers and targets to manage metastatic spread and improve outcomes for patients with OS.

Up-regulation of pro-angiogenic molecules and events does not relate with an angiogenic switch in metastatic osteosarcoma cells but to cell survival features.

Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children. Metastases represent a major clinical challenge and an estimated 80% would present undetectable micrometastases at diagnosis. The identification of metastatic traits and molecules would impact in micrometastasis management. We demonstrated that OS LM7 metastatic cells secretome was able to induce microvascular endothelium cell rearrangements, an angiogenic-related trait. A proteomic analysis indicated a gain in angiogenic-related pathways in these cells, as compared to their parental-non-metastatic OS SAOS2 cells counterpart. Further, factors with proangiogenic functions like VEGF and PDGF were upregulated in LM7 cells. However, no differential angiogenic response was induced by LM7 cells in vivo. Regulation of the Fas-FasL axis is key for OS cells to colonize the lungs in this model. Analysis of the proteomic data with emphasis in apoptosis pathways and related processes revealed that the percentage of genes associated with those, presented similar levels in SAOS2 and LM7 cells. Further, the balance of expression levels of proteins with pro- and antiapoptotic functions in both cell types was subtle. Interestingly and of relevance to the model, Fas associated Factor 1 (FAF1), which participates in Fas signaling, was present in LM7 cells and was not detected in SAOS2 cells. The subtle differences in apoptosis-related events and molecules, together with the reported cell-survival functions of the identified angiogenic factors and the increased survival features that we observed in LM7 cells, suggest that the gain in angiogenesis-related pathways in metastatic OS cells would relate to a prosurvival switch rather to an angiogenic switch as an advantage feature to colonize the lungs. OS metastatic cells also displayed higher adhesion towards microvascular endothelium cells suggesting an advantage for tissue colonization. A gain in angiogenesis pathways and molecules does not result in major angiogenic potential. Together, our results suggest that metastatic OS cells would elicit signaling associated to a prosurvival phenotype, allowing homing into the hostile site for metastasis. During the gain of metastatic traits process, cell populations displaying higher adhesive ability to microvascular endothelium, negative regulation of the Fas-FasL axis in the lung parenchyma and a prosurvival switch, would be selected. This opens a new scenario where antiangiogenic treatments would affect cell survival rather than angiogenesis, and provides a molecular panel of expression that may help in distinguishing OS cells with different metastatic potential.

Acquisition of stem associated-features on metastatic osteosarcoma cells and their functional effects on mesenchymal stem cells.

BACKGROUND: Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children and young adults. Metastases are a major clinical challenge in OS. In this context, 20% of OS patients are diagnosed with metastatic OS, but near 80% of all OS patients could present non-detectable micrometastases at the moment of diagnosis. METHODS: Osteogenic differentiation; doxorubicin exclusion assay; fluorescence microscopy; RT-qPCR; proteomic analysis. RESULTS: Our results suggest that metastatic OS cells possess a diminished osteoblastic differentiation potential with a gain of metastatic traits like the capacity to modify intracellular localization of chemodrugs and higher levels of expression of stemness-related genes. On the opposite hand, non-metastatic OS cells possess bone-associated traits like higher osteoblastic differentiation and also an osteoblastic-inducer secretome. OS cells also differ in the nature of their interaction with mesenchymal stem cells (MSCs), with opposites impacts on MSCs phenotype and behavior. CONCLUSIONS: All this suggests that a major trait acquired by metastatic cells is a switch into a stem-like state that could favor its survival in the pulmonary niche, opening new possibilities for personalized chemotherapeutic schemes. GENERAL SIGNIFICANCE: Our work provides new insights regarding differences among metastatic and non-metastatic OS cells, with particular emphasis on differentiation potential, multidrug resistance and interaction with MSCs.

Metastatic Niches and the Modulatory Contribution of Mesenchymal Stem Cells and Its Exosomes.

Mesenchymal stem cells (MSCs) represent an interesting population due to their capacity to release a variety of cytokines, chemokines, and growth factors, and due to their motile nature and homing ability. MSCs can be isolated from different sources, like adipose tissue or bone marrow, and have the capacity to differentiate, both in vivo and in vitro, into adipocytes, chondrocytes, and osteoblasts, making them even more interesting in the regenerative medicine field. Tumor associated stroma has been recognized as a key element in tumor progression, necessary for the biological success of the tumor, and MSCs represent a functionally fundamental part of this associated stroma. Exosomes represent one of the dominant signaling pathways within the tumor microenvironment. Their biology raises high interest, with implications in different biological processes involved in cancer progression, such as the formation of the pre-metastatic niche. This is critical during the metastatic cascade, given that it is the formation of a permissive context that would allow metastatic tumor cells survival within the new environment. In this context, we explored the role of exosomes, particularly MSCs-derived exosomes as direct or indirect modulators. All this points out a possible new tool useful for designing better treatment and detection strategies for metastatic progression, including the management of chemoresistance.